Induced pluripotent stem (iPS) cells have enormous potential for the repair of diseased or traumatized blood vessels. Indeed, we, among the first, have successfully induced iPS cell differentiation to smooth muscle cells (SMC) using all-trans retinoid acid (atRA). Our long-term goal is to regenerate functional human blood vessels using patient-derived iPS cells. The key to a functional blood vessel regeneration using iPS cells is the differentiation and maintenance of the contractile phenotype of the vascular SMC. The overall hypothesis is that the coordination of the key signaling molecules with the biomimetic microenvironment defined by an advanced scaffold is required for achieving the contractile phenotype of iPS-derived vascular SMC and functional blood vessel regeneration. The Hippo-YAP signaling pathway has recently been found to play a critical role in maintaining iPS cell pluoripotency. Supported by preliminary data, we hypothesize that Yap1 is a critical molecule inhibiting the differentiation of iPS cells to vascular SMC and suppressing the contractile phenotype of vascular SMC. We developed 3D porous and nanofibrous (NF) scaffolds and found that the NF architecture enhanced iPS cell differentiation to vascular SMC and the contractile phenotype over control scaffolds. In this project, we will first define the role of Yap in regulating iPS cell differentiation to vascular SMC and vascular SMC phenotypic switch in a 2D culture system. We will then develop optimal NF scaffolds to define the role of Yap1 in regulating iPS cell differentiation to vascular SMC in 3D culture system. We will also develop controlled release system inside the scaffolds to maximize the utility of atRA along with Yap1 modulation in enhancing the vascular SMC differentiation and their mature contractile phenotype maintenance. Built on these mechanistic understandings and advanced technologies, we will engineer blood vessels and evaluate them using bioreactors and a rat implantation model. By accomplishing these specific aims, we will improve mechanistic understandings of iPS cell differentiation to vascular SMC and develop key technologies to advance the therapeutic utility of patient based iPS cells for human vascular regeneration.